Genes on the same genomic position but on the different alleles can be different in expression level. This phenomenon is a relatively new concept that has been reported recently (Knight J C. Allele-specific gene expression uncovered, Trends Genet. March; 20(3): 113-6. PMID: 15049300, 2004).
Genes expressed differentially between alleles are roughly classified into two types: imprinted gene and non-imprinted gene. In the phenomenon of the former, i.e. the imprinted gene, when one allele is inherited from one of the parents while the other allele from the other parent, one of the alleles is physiologically inactivated (e.g., methylated), whereby the expression of the gene is inhibited in cells or tissues. Also for the latter (i.e. non-imprinted genes), there are some cases where a difference in expression level is observed between alleles. With respect to such a difference, it is thought that a genomic polymorphism in a gene or a region adjacent thereto between alleles serves as a cis-acting element for regulating the expression of a gene in the vicinity of the polymorphism, thereby leading to a difference in gene expression level between alleles. Variations in expression of each allele resulting from different genomic DNA sequences are considered to be properties that are inherited over generations, and such properties may influence differences in gene expression levels among individuals and differences in body constitutions, pathological conditions and risks thereof, and responsiveness to drugs, among individuals.
Difference in gene expression levels between alleles can be most accurately assayed within a same cell, or under identical environmental conditions. In assaying a difference in gene expression levels between alleles, it is important from which allele a certain RNA is derived can be determined. To this end, the presence of a polymorphism that enables distinction of alleles (e.g., SNP) is required in an RNA sequence, which is a transcript of a gene, and the polymorphism in the RNA sequence is measured to determine a difference in gene expression level between alleles. There are several reports on determining a difference in gene expression level between alleles using such a polymorphism (SNP) on RNA (Cowles C R, Hirschhorn J N, Altshuler D, Lander E S, Detection of regulatory variation in mouse genes, Nat Genet. November; 32(3): 432-7, PMID, 12410233, 2002; Yan H, Yuan W, Velculescu V E, Vogelstein B, Kinzler K W, Related Allelic variation in human gene expression, Science. August 16; 297 (5584): 1143, PMID, 12183620, 2002; Bray N J, Buckland P R, Owen M J, O'Donovan M C, Cis-acting variation in the expression of a high proportion of genes in human brain, Hum Genet., 2003 July; 113 (2): 149-53. Epub, May 1, PMID: 12728311, 2003).
However, the techniques employed in the reports are a combination of RT-PCR with a direct sequencing reaction or single-nucleotide extension, wherein cDNA is synthesized from mRNA and amplified, and then arbitrarily selected polymorphisms are individually subjected to typing. These techniques are not capable of simultaneously measuring many genes.
To date, extensive analysis of many genes using microarrays for SNP typing has been reported (Lo H S, Wang Z, Hu Y, Yang H H, Gere S, Buetow K H, Lee M P, Allelic variation in gene expression is common in the human genome, Genome Res. August; 13(8): 1855-62. PMID: 12902379, 2003). In this analysis, mRNA with poly(A) is converted into cDNA by the common RT method using a poly(T) primer, samples are prepared by the multiplex PCR technique using many specific primers in accordance with the same protocol as a conventional genomic DNA typing technique, and samples are hybridized to the arrays to measure the expression levels of cDNA (mRNA) that differ between alleles based on the signal ratio. However, mature mRNA with poly (A) has only exon sequences following splicing, and so such sequences are too short to comprise enough polymorphisms (SNPs) to be evaluated. Thus, because available polymorphisms (SNPs) are limited, it is difficult to find a gene whose expression level varies in every allele.
The correlation between genetic polymorphism and certain phenotype and gene expression (e.g., difference in disease or drug efficacy) has drawn attention. However, to study the correlation between certain genetic polymorphism and phenotype and gene expression, it is required to examine a huge number of SNPs for respective traits in the case of, for example, genomic SNPs (about 10 million according to the NCBI dbSNP (build 123) reported in October 2004), and thus it is indeed difficult to do so.
If genes whose phenotype and gene expression differs between alleles can be rapidly and effectively selected in order to study the correlation between the thus selected genetic polymorphism and the phenotype and gene expression, accordingly, the cause of a disease, effective therapeutic methods, or the like may be examined by the completed procedures.